Kinetics of green solid-liquid extraction of andrographolide from Andrographis paniculata: effects of particle size and solid-liquid ratio

Symbols

Ŷ

Overall yield of desired product extract

Ŝ_D

Overall selectivity of desired product extract

Ŝ_U

Overall selectivity of undesired product extracts (other phytochemical compounds)

Ŝ_D/U

Overall selectivity of desired product extract with respect to undesired product extracts (other phytochemical compounds)

A_D

Amount of desired extract (andrographolide or whole extracts)

A_SM

Amount of solid material (Andrographis paniculata)

A_AASM

Actual amount of solid material disappeared in extraction

C_Bt

Concentration of andrographolide in the liquid extracts at any time, t (min), mg l^-1

C_Be

Equilibrium concentration of andrographolide in the liquid extracts at saturation with insoluble solids, mg l^-1

k_obs

Observed first-order or second-order specific extraction rate constant, min^-1 and l mg^-1 min^-1, respectively

r_B0

Initial rate of extraction for andrographolide, mg l^-1 min^-1

t

Extraction time, min

1 Introduction

Andrographis paniculata (AP) is identified as one of the commercially important medicinal plants. It is one of about 40 species belonging to Andrographis, which is a genus within the Acanthaceae family, with a reputation in traditional medicine [1]. AP is a well-known herb that has been successfully used for several centuries against a wide range of diseases in East Asia, South Central Asia and South East Asia [2, 3]. The major medicinally active constituent of AP was found to be andrographolide diterpenoid lactone [4, 5], with a molecular structure as shown in Figure 1, molecular weight of 350.45 g mol^-1 and molecular formula C₂₀H₃₀O₅. Andrographolide is typically produced from AP through solid-liquid extraction. There are numerous published studies relating to the pharmacology, bioactivity and phytochemical isolation of andrographolide and other phytochemical compounds from AP. Some of the studies are dedicated to the extraction of andrographolide from AP [6–14]. However, only one report studied the kinetics of solid-liquid extraction of andrographolide [9]. The cost of purified andrographolide extract and its derivatives could be around US$100,000/kg as reported by chemical suppliers [3, 15].

Figure 1:

Molecular structure of andrographolide.

Many efforts have been made to describe the kinetics of solid-liquid extraction for distinct types of phytochemical compound(s) based on the step consideration of either solute diffusion or dissolution of solute from different plant materials, as the rate-determining step. Fick’s second law of diffusion or the rate laws derived from the concept of chemical reaction kinetics are respectively employed. Although most of the reported studies used the Fickian second law of diffusion [3, 9, 16–24], very few published works utilized the rate laws [25–29]. Chan et al. [30] reported a good review on the modeling and kinetics study of conventional and assisted batch solvent extraction using both techniques. However, this study focused on the application of rate laws for the analysis of extraction kinetics for the green dissolution of andrographolide step of the solid-liquid extraction.

Solid-liquid extraction is a time-dependent process [31]. Extraction kinetics is concerned with the measurement and interpretation of extraction rates. A successful profitable operation requires proper sizing of solid-liquid extractor and understanding of the extraction kinetics. The kinetic parameters such as specific extraction rate constant and order of extraction play an essential role in product and process development [32]. They are needed to characterize the solid-liquid extraction, use in the identification of rate-controlling step of the process, predictive modeling, design and scale-up to a pilot and commercial size of any solid-liquid extractor.

Green solid-liquid extraction is an approach to the processing techniques of extraction through the use of non-toxic solvents that eliminate or reduce generation of products and byproducts that are hazardous to human health and the environment. This is similar to the definition of green extraction given by Chemat et al. [33], and the general definition of green chemistry proposed by Anastas [34]. Generally, when selecting a solvent for extraction, the rule of thumb is that “like dissolves like” [35], thus, this means that polar compounds are dissolved by polar solvents, and non-polar compounds are dissolved by non-polar solvents. Andrographolide is a polar compound because of the presence of hydroxyl and carbonyl groups attached to the rings. The solubility of andrographolide in water was the least when compared with other members of polar protic solvents such as methanol, ethanol, butanol and propanol at lower temperatures [36]. Nevertheless, most of the volatile polar solvents are not completely free from toxicity, irritation, and flammability [37]. Supercritical carbon dioxide is widely regarded as green. Even so, it is a very low polarity compound [38], behaves more like non-polar solvents [37, 39] and needs a volatile protic co-solvent as a modifier for the effective extraction of polar compounds such as andrographolide [37, 39–42].

However, two of the 12 principles of green chemistry proposed by Anastas and Warner [43] necessitate the use of safer solvents and auxiliaries, and less or nonhazardous chemicals that will not generate toxic substances in the product and process synthesis. In addition, the second principle of the six principles of green extraction proposed by Chemat et al. [33] requires the use of alternative solvents and principally water or agro-solvents. This work has chosen water over other members of polar protic, aprotic and supercritical carbon dioxide solvents to be the best green solvent. Water is considered to be most likely the greenest alternative solvent because of its unique qualities such as non-toxicity, non-flammability, being odorless, being colorless, availability, renewability, low cost, and other tunable properties such as boiling point, dielectric constant, dipole moment and Kamlet–Taft solvatochromic parameters [37, 44–46]. To the best of our knowledge, very little is known and published on the kinetics of green solid-liquid extraction for andrographolide from AP using water. This paper presents the kinetics, characterizes the green solid-liquid extraction of andrographolide and finds the influences of particle size and liquid-solid ratio on the concentration-dependent term and specific extraction rate constant of the extraction rate law.

2 Materials and methods

2.2 Kinetics measurements

In order to obtain kinetic data from the system wherein the effects of diffusion limitations are not significant, the experiments of green solid-liquid extraction for andrographolide from AP were carried out in a pressurized liquid extractor similar with the following [34, 47–49], by employing Dionex ASE 100®, USA as shown in Figure 2. The distilled water was used as the solvent. The experiments were performed under isothermal conditions at 80°C to find the influences of particle size and solid-liquid ratio on the concentration-dependent terms and specific extraction rate constant of the kinetic expression at various times. Five different classes of average particle sizes of solid material, namely, 1.200 mm, 0.855 mm, 0.605 mm, 0.375 mm and 0.175 mm were used during the study, each for the time of 5 min, 10 min, 15 min, 20 min and 25 min, and for the solid-liquid ratio of 1/10 and 1/50 g/ml.

Figure 2:

Schematic diagram of the pressurized liquid extraction (PLE) system.

A 10 g sample of 1.200 mm average particle size was packed into a 100 ml extraction cell (to have a solid-liquid ratio of 1:10 g ml^-1). The ASE 100 was programmed to the set point temperature of 80°C, static extraction time of 5 min, purge time of 60 s and flushing volume of 30%. When the system was under steady state and the oven was ready, the extraction cell was placed on the cell holder, and the cell door was closed to allow the cell to be inside the oven. Then, filling of the extraction cell commenced by moving the solvent via a fast pump from the solvent bottle into the cell. The extraction was started at a pressure of 1300 psi. The pressure of the system kept increasing up to around 1600 psi. After completion of the process, compressed nitrogen gas and the fixed volume of fresh solvent for flushing moved all the extract into the collection vial. The ASE 100® was reprogrammed according to specified values for each of the remaining experimental runs, and the above procedure was repeated.

2.3 Analysis of AP green extracts

2.3.1 Phytochemical screening of AP green extracts:

The general tests for preliminary phytochemical screening of water extracts of AP were performed to identify its phytochemical constituents. These include the various types of tests for carbohydrates of primary metabolites: free reducing sugars, cardiac glycosidase, tannins, flavonoids, terpenes/steroidal rings, alkaloids, saponins and anthraquinone derivatives of secondary metabolites.

2.3.2 HPLC analysis of andrographolide:

The identification and quantification of andrographolide in the water extracts were carried out by using a Waters 2690 Alliance HPLC equipped with Waters 996 photodiode detector absorbance, USA, connected to a reversed-phase column (Lichrospher 100 RP-18, 5 μm, 250 mm×4.6 mm, Germany). A stock solution (1000 ppm) was prepared from the standard chemical of andrographolide. Each sample was filtered with a Whatman nylon syringe, and then transferred into a 1.5 ml HPLC vial. The injection volume was 20 μl with the aid of an autosampler. The detection of andrographolide concentration was measured at 223 nm using the isocratic gradient method [50] at the ratio of methanol/water (60:40) as the mobile phase, constant flow rate of 0.7 ml/min and 23°C. Chromatographic peaks of andrographolide were identified by comparing the retention time with that of the standard chemical. Figure 3 depicts one of the typical HPLC chromatograms obtained from AP water extracts.

Figure 3:

High-performance liquid chromatography (HPLC) chromatogram of Andrographis paniculata (AP) water extracts.

The quantification for andrographolide in the water extracts was analyzed using a calibration curve of the standard with an accuracy of 99.8%. The overall yield of andrographolide and extracts were calculated from Eq. (1):

(1)Y^(%)=ADASM×100 (1)

where A_D and A_SM are the amounts of desired extract (andrographolide), and solid material (AP) used in the solid-liquid extraction. The overall selectivity of andrographolide (Ŝ_D) and overall selectivity of andrographolide with respect to other phytochemical compounds (Ŝ_D/U) were estimated according to the new equations reported by Isah et al. [51] as shown in Eqs. (2) and (3), respectively, where A_AASM and S^U are the actual amount of solid material disappeared in the solid-liquid extraction and the overall selectivity of undesired extracts (other phytochemical compounds), respectively:

(2)S^D(%)=ADAAASM×100 (2)

(3)S^D/U(%)=S^DS^U×100 (3)

2.4 Kinetics of solid-liquid extraction

In the solid-liquid extraction of plant material, soluble phytochemical component(s) are dissolved from the surface and inside the porous particles of plant cellular material by liquid (i.e. solvent). The mechanism for the solid-liquid extraction of phytochemical compound(s) consists of the following steps:

1. The solvent must be transferred onto the external surface of the porous solid particle from the bulk liquid solvent (diffusion).

2. The solvent penetrates or diffuses into the porous solid particle (intraparticle diffusion).

3. The solute dissolution (phase change of solute as it dissolves into the solvent) to form solution possibly by physical or chemical reaction.

4. The solution diffuses through the mixture in the porous particle to the external surface of the solid (intraparticle diffusion).

5. The solution is transferred to the bulk solvent from the external surface of the solid (diffusion).

The scheme illustrated in Figure 4 was chosen as the best scheme representing the sequences of multiple competing green solid-liquid extractions of andrographolide with other phytochemical compounds from AP in a series with multiple transformations and reactions of andrographolide and other phytochemical compound (s) in parallel or a series from the new schemes proposed for solid-liquid extraction by Isah et al. [51].

Figure 4:

Multiple competing green solid-liquid extractions in series with multiple reactions of andrographolide (desired product extract) from Andrographis paniculata (AP) and other phytochemical compounds (undesired products) in parallel and series.

The letters in the scheme represent the following: A–AP, S–green solvent (water), B–andrographolide (desired product extract), undesired product extracts (C–apigenin, D–glucose, E–7-O-methyl wogonin, F–skullcapflavone I, G–andrographidines A, B, C, D, E and F; and H–unknown competing product extracts in relatively larger amounts from free reducing sugars, cardiac glycosidase, tannins, alkaloids, saponins and anthraquinone derivatives), undesired products of andrographolide reactions (I–isoandrographolide, J–14-deoxyandrographolide, K–deoxyandrographolide, L–andrograpanin, P–neoandrographolide, Q–andrographiside, R–14-deoxy-11, 12-didehydroandrographolide, T–dehydroandrographolide, U–14- deoxy-11-hydroxyandrographolide and V–14-deoxy-12-hydroxyandrographolide).

At equilibrium under ideal conditions, all solutes (i.e. soluble phytochemical compounds) are assumed to be dissolved by liquid solvent [52, 53]. The concentration of andrographolide is assumed to be uniform within the mixture. In addition, the concentration of andrographolide in the liquid phase (i.e. solution) is equal to the concentration of andrographolide retained in the solid phase (i.e. insoluble solids). The first-order kinetic expression for the dissolution of andrographolide can be written in differential form based on the concentration of andrographolide at any time and the equilibrium concentration of andrographolide in the liquid extracts at saturation with insoluble solids as shown in Eq. (4) [54–64]:

(4)dCBtdt=kobs(CBe-CBt) (4)

where C_Bt (mg l^-1) is the concentration of andrographolide in the liquid extracts at any time, t (min), C_Be (mg l^-1) is the equilibrium concentration of andrographolide in the liquid extracts at saturation with insoluble solids and k_obs (min^-1) is the observed first-order specific extraction rate constant of the kinetic model.

Integrating Eq. (4) with the boundary conditions of C_Bt =0 at t=0 and C_Bt =C_Bt at t=t gives Eq. (5):

(5)ln(CBeCBe-CBt)=kobst (5)

and

(6)CBt=CBe(1-e-kobst) (6)

Rearranging Eq. (5) into a linear form gives Eq. (7):

(7)log(CBe-CBt)=logCBe-kobs2.303t (7)

It is noted that k_obs and C_Be can be determined from the slope and intercept of the plots of log (C_Be-C_Bt) against t for different experimental conditions as shown in Eq. (7).

Similarly, the second-order rate law for the dissolution of andrographolide can be written in differential form based on the concentration of andrographolide at any time and the equilibrium concentration of andrographolide in the liquid extracts at saturation with insoluble solids as represented in Eq. (8) [54–65]:

(8)dCBtdt=kobs(CBe-CBt)2 (8)

where k_obs (l mg^-1 min^-1) is the observed second-order specific extraction rate constant of the kinetic expression. Integrating Eq. (8) with the boundary conditions of C_Bt =0 at t=0 and C_Bt =C_Bt at t=t gives Eq. (9):

(9)1(CBe-CBt)-1CBe=kobst (9)

Rearranging Eq. (9) gives Eq. (10):

(10)CBt=CBe2kobst1+CBekobst (10)

Rearranging Eq. (10) into a linear form gives Eq. (11):

(11)tCBt=1kobsCBe2+tCBe (11)

From Eq. (9), it is noted that as t approaches 0, the initial rate of extraction for andrographolide can be represented as shown in Eq. (12):

(12)rB0=kobsCBe2 (12)

The initial rate of extraction for andrographolide r_B0 (mg l^-1 min^-1), k_obs and C_Be can be evaluated experimentally from the intercept and the slope of the plots of t/C_Bt against t for different experimental conditions as illustrated in Eqs. (11) and (12).

2.4.1 Statistical analysis:

For the statistical inferences of the estimated kinetic parameters, the standard errors, Pearson’s r, and adjusted R² were used in the analysis. Standard error (σ_ki) of the kinetic parameter i estimate can be obtained from Eqs. (13) or (14) as the square root of the corresponding diagonal element of the inverse of matrix A multiplied by standard deviation (σ) [66, 67]. It is used to indicate the uncertainty around the estimate of the mean value. It depends on both the standard deviation and the sample size:

(13)σki=σ(A-1)ii (13)

(14)σki(Y^0)=est.σ(Y^0)=σ(1n+(X0-X)2∑(Xi-X)2)12 (14)

Pearson’s r is a correlation coefficient (ρ) that measures the linear correlation for two variables Y and X between -1 and +1. It indicates negative correlation when the value is -1, no correlation and positive correlation when the value is 0, and +1, respectively. This can be obtained as shown in Eq. (15) as the covariance of the two variables divided by the product of their standard deviations [68]:

(15)ρX,Y=cov(X, Y)σXσY (15)

Adjusted R² is a statistical parameter that adjusted the degrees of freedom of the two quantities when showing the goodness of model fit. It can be used to compare equations fitted not only to a specific set of data, but also to two or more entirely different sets of data. This can be determined as shown in Eq. 16 [67]:

(16)Adj.R2=1-(RSSn-p)/(n-p)(CTSS)/(n-1)=1-(1-R2)(n-1n-p) (16)

where R² is given in Eq. (17), RSS_n-p is the corresponding residual sum of squares, and CTSS denotes the corrected total sums of squares:

(17)R2=1-RSSn-pCTSS (17)

3 Results and discussion

Table 1 shows the results of phytochemical screening of AP water extracts obtained from AP in the green solid-liquid extraction of andrographolide at 80°C. It can be seen that the water extracts contain various phytochemical compounds. These include carbohydrates of primary metabolites: free reducing sugars, cardiac glycosidase, tannins, flavonoids, terpenes/steroidal rings, alkaloids, saponins and anthraquinone derivatives of secondary metabolites.

Figures 5–9 display the effects of various particle sizes on the overall yield of extracts, overall yield of andrographolide, overall selectivity of andrographolide, overall selectivity of andrographolide with respect to other phytochemical compounds and rate of andrographolide extraction in green solid-liquid extraction of andrographolide from AP at 80°C for different solid-liquid ratios, respectively. Likewise, the influences of different particle sizes and solid-liquid ratios on the rate of green solid-liquid extraction of andrographolide from AP, and the fitting of the kinetics using the first-order and second-order extraction rate laws are depicted in Figures 10 and 11, respectively.

Figure 5:

Effect of particle size on the overall yield of extracts from Andrographis paniculata (AP) in the green solid-liquid extraction of andrographolide at 80°C for solid-liquid ratio of (A) 1:10 (g ml^-1) and (B) 1:50 (g ml^-1).

Figure 6:

Effect of particle size on the overall yield of andrographolide from Andrographis paniculata (AP) in the green solid-liquid extraction of andrographolide at 80°C for solid-liquid ratio of (A) 1:10 (g ml^-1) and (B) 1:50 (g ml^-1).

Figure 7:

Effect of particle size on the overall selectivity of andrographolide from Andrographis paniculata (AP) in the green solid-liquid extraction at 80°C for solid-liquid ratio of (A) 1:10 (g ml^-1) and (B) 1:50 (g ml^-1).

Figure 8:

Effect of particle size on the overall selectivity of andrographolide with respect to other phytochemical compounds from Andrographis paniculata (AP) in the green solid-liquid extraction at 80°C for solid-liquid ratio of (A) 1:10 (g ml^-1) and (B) 1:50 (g ml^-1).

Figure 9:

Effect of particle size on the rate of solid-liquid extraction of andrographolide from Andrographis paniculata (AP) in the green solid-liquid extraction at 80°C for solid-liquid ratio of (A) 1:10 (g ml^-1) and (B) 1:50 (g ml^-1).

Figure 10:

First-order kinetic model testing for the green solid-liquid extraction of andrographolide from Andrographis paniculata (AP) at 80°C for different particle sizes and solid-liquid ratios of (A) 1:10 (g ml^-1) and (B) 1:50 (g ml^-1).

Figure 11:

Second-order kinetic model testing for for the green solid-liquid extraction of andrographolide from Andrographis paniculata (AP) at 80°C for different particle sizes and solid-liquid ratios of (A) 1:10 (g ml^-1) and (B) 1:50 (g ml^-1).

The investigation covers five distinct average particle sizes ranging from 0.175 mm to 1.20 mm for different solid-liquid ratios of 1:10 and 1:50 g ml^-1. Figures 5A, B, 6A, B, 7A, B, 8A, B, 9A and B compare the effect of average particle sizes of AP and extraction time on the overall yield of water extracts, overall yield of andrographolide, overall selectivity of andrographolide, overall selectivity of andrographolide with respect to other phytochemical compounds, and the rate of andrographolide extracted under isothermal conditions at 80°C, respectively.

The maximum overall yields of water extracts, overall yield of andrographolide, overall selectivity of andrographolide, overall selectivity of andrographolide with respect to other phytochemical compounds and amount of andrographolide produced can be seen from the Figures to be 23.73% and 21.20%, 4.11%, and 5.59%, 17.30% and 26.37%, 20.92% and 35%, and 410.55 mg ml^-1 and 111.83 mg ml^-1 at 20 min and 15 min, respectively. Similarly, the minimum overall yields of water extracts, overall yield of andrographolide, overall selectivity of andrographolide, overall selectivity of andrographolide with respect to other phytochemical compounds and amount of andrographolide produced can be observed from the same Figures to be 15.33% and 12.06%, 1.55% and 1.30%, 10.10% and 10.82%, and 11.23% and 12.13% at 5 min for average particle size of 1.200 mm, respectively. It can also be seen that the plots of the amount of andrographolide water extract produced, and overall yields of andrographolide from all the plots follow the pattern of exponential growth or law of diminishing returns. The concentrations of andrographolide kept increasing up to a different time for each class of particle size where andrographolide reached its equilibrium or maximum concentrations. Then, the concentration started to decline except in the cases for the average particle sizes of 1.200 mm, 0.855 mm and 0.605 mm as shown in Figures 5A, 6A, 7A, 8A and 9A; 1.200 mm and 0.855 mm as presented in Figures 5B, 6B, 7B, 8B and 9B, where the concentrations of andrographolide continue to increase.

Based on the results discussed, it can be deduced that the green solid-liquid extraction of andrographolide from AP under isothermal conditions at 80°C is not feasible after reaching 20 min for the average particle sizes of 1.200 mm, 0.855 mm, 0.605 mm and the solid-liquid ratio of 1:50 g ml^-1. Similarly, the process is not profitable after reaching 15 min for the average particle diameter of 0.375 mm, 0.175 mm, when the solid-liquid ratio is 1:50 g ml^-1. However, in the case where the ratio of solid-liquid is 1:10 g ml^-1, the results suggest that it is feasible to continue with the process after reaching 25 min and 20 min with average particle sizes of solid material of 1.200 mm, 0.855 mm, 0.605 mm; and 0.375 mm, and 0.175 mm, respectively. It can be inferred from the investigations that as the particle size of AP decreases, the rate of solid-liquid extraction of andrographolide increases, which will consequently affect the specific extraction rate constant.

At the first attempt to describe the kinetics of solid-liquid extraction, a first-order extraction in terms of andrographolide dissolution was assumed to be uniformly distributed throughout the porous solid particles. The kinetic data were fitted with first-order rate equation as exhibited in Figure 10A and B. However, the straight lines in the plot of log(C_Be-C_Bt) against t did not show a good agreement with the experimental data with the first-order kinetic model for the different average particle sizes of the solid material and solid-liquid ratio. Consequently, the first-order was substituted with a second-order and the assumption of extraction in terms of dissolution of andrographolide was held. The fitting of the experimental data with the second-order kinetic model can be seen in Figure 11A and B. It can be observed from the plots of t/C_Bt versus t that the linear fittings show a better agreement of the kinetics data with the second-order kinetic model for the different classes of average particle sizes and solid-liquid ratios. The straight lines provided further evidence that the assumption of the second-order extraction was valid. This can be confirmed from the results shown in Tables 4 and 5 for the second-order model, with higher Pearson’s r, and adjusted R² values between (0.968 and 1.000), (0.904 and 0.999), and (0.985 and 0.999), (0.974 and 0.999) than those presented in Tables 1 and 2 of the first-order model, between (-0.878 and -0.971), (0.655 and 0.914), (-0.895 and -1.000) and (0.700 and 0.999), respectively. The uncertainty around the estimate for the slope and intercept were calculated in the form of standard error as reported in Englezos and Kalogerakis [66].

The kinetic parameters k_obs, C_Be, and r_B0 of the first-order and second-order kinetics models were evaluated from the slope and intercept of the plots with their precisions. The values found with their precisions in the form of standard errors, Pearson’s correlation coefficients and adjusted coefficient of determination are presented in Tables 2–5. Therefore, second-order rate law of extraction satisfactorily fits the kinetic data. It can be inferred from the results obtained that as the average particle size of solid material decreases and solid-liquid ratio increases, the observed specific extraction rate constant, k_obs and the initial rate of solid-liquid extraction of andrographolide, r_B0 increase. It was found that k_obs and r_B0 increased almost twofold and fivefold when the particle size was decreased from 1.200 mm to 0.175 mm, while k_obs and r_B0 increased from fourfold to fivefold and from nearly threefold to more than sixfold when the solid-liquid ratio increased from 1:10 to 1:50 g ml^-1, respectively.

4 Conclusions

The influences of particle size and solid-liquid ratio on the kinetics of green solid-liquid extraction for andrographolide from AP were investigated. The extraction kinetics was found to follow second-order rate law. From the results, it can be deduced that as the particle size of solid material decreases and the solid-liquid ratio increases, the observed specific extraction rate constant, k_obs, and the initial rate of solid-liquid extraction of andrographolide, r_B0 were significantly increased. It was found that k_obs increased to nearly twofold from 6.53×10^-4 to 1.28×10^-3 l mg^-1 min^-1 for a solid-liquid ratio of 1:10 g ml^-1 when the particle size was decreased from 1.200 mm to 0.175 mm. Moreover, k_obs increased over fivefold, 3.44×10^-3 l mg^-1 min^-1 and virtually fourfold, 4.96×10^-3 l mg^-1 min^-1 for particle sizes of 1.200 mm and 0.175 mm with overall rise of more than sevenfold when the solid-liquid ratio was increased to 1:50 g ml^-1, respectively.